Listeria monocytogenes is a Gram positive facultative intracellular bacterium that is capable of invading a wide range of host cell types and replicating in the cytosol. Proteins which mediate invasion, vacuolar escape, and survival in the host cytosol are all transcriptionally controlled by the positive regulatory factor PrfA. PrfA belongs t the cAMP receptor protein (Crp) family of transcription factors, which are activated upon binding small molecule cofactors. To date, a cofactor has not been identified for PrfA, although it is believed that a signal(s) specific to the host cell cytosol triggers PrfA activation, leading to virulence gene expression. We performed a forward genetic screen to identify factors that allow Lm to recognize its cytosolic localization and activate transcription of virulence genes. Our screen for 'cytosol sensing' was extremely successful, as we identified many genes that have severe defects in PrfA-dependent gene regulation, but have never before been associated with virulence. Two transposon mutants were chosen for further investigation, and each displayed a 3-5 log defect in colonization of the spleens and livers of intravenously infected mice. Experiments detailed in this application aim to dissect how these mutations regulate virulence gene expression in Listeria.